van der Wees J et al. (1998), Inhibition of retinoic acid receptor-mediated s...

XB-ART-15634

Development 1998 Feb 01;1253:545-56. doi: 10.1242/dev.125.3.545.

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Inhibition of retinoic acid receptor-mediated signalling alters positional identity in the developing hindbrain.

van der Wees J , Schilthuis JG , Koster CH , Diesveld-Schipper H , Folkers GE , van der Saag PT , Dawson MI , Shudo K , van der Burg B , Durston AJ .

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Retinoids regulate gene expression via nuclear retinoic acid receptors, the RARs and RXRs. To investigate the functions of retinoid receptors during early neural development, we expressed a dominant negative RARbeta in early Xenopus embryos. We obtained evidence that dominant negative RARbeta specifically inhibits RAR/RXR heterodimer-mediated, but not RXR homodimer-mediated, transactivation. Both all-trans- and 9-cis-RA-induced teratogenesis were, however, efficiently opposed by ectopic expression of dominant negative RARbeta, indicating that only RAR/RXR transactivation is required for retinoid teratogenesis by each of these ligands. Experiments with two RXR-selective ligands confirmed that activation of RXR homodimers does not cause retinoid teratogenesis. Dominant negative RARbeta thus specifically interferes with the retinoid signalling pathway that is responsible for retinoid teratogenesis. Dominant negative RARbeta-expressing embryos had a specific developmental phenotype leading to disorganization of the hindbrain. Mauthner cell multiplications in the posterior hindbrain, and (both anteriorly and posteriorly) expanded Krox-20 expression domains indicated (partial) transformation of a large part of the hindbrain into (at least partial) rhombomere 3, 4 and/or 5 identity. In contrast, the fore- and midbrain and spinal cord appeared to be less affected. These data indicate that RARs play a role in patterning the hindbrain.

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Species referenced: Xenopus
Genes referenced: egr2 en2 hoxb3 lgals4.2 lsamp otx2 rab40b rarb tnfrsf10b
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	Fig. 1. DN RARb expression inhibits ATRA-induced transcription of a reporter construct containing a DR5 RARE in its natural context, in Xenopus embryos. Embryos were injected with 1.75 ng DN RARb RNA and/or 165 pg reporter (-1470+156Luc; RARb promoter) DNA (‘DN RARb’ or ‘no RNA’, respectively). At stage 10, embryos were transferred to a medium containing 3´10-7, or 10-6 M ATRA, or no additive. After 7-hours incubation, luciferase activity was determined. Each bar represents the mean value of at least 3 pools of 5 embryos ± s.e.m.. Inhibition by DN RARb of activation of the reporter by ATRA was observed in every reporter experiment performed.
	Fig. 2. Effects of DN RARb expression on normal or ATRA-disturbed Xenopus development. DN RARb reduces ATRA teratogenesis. (A) DN RARb expression (top embryo) causes no prominent anteroposterior defects compared with control embryos, but experimental embryos are clearly smaller. (B,C) Teratogenic effects of a 14-hour treatment with 10-6 M ATRA are much less severe in DN RARb-expressing embryos (C) than in controls (B). Almost complete rescue from this high dose of ATRA is possible (C, bottom). (D) Wild-type Otx-2 expression pattern in control stage-21 embryo. (E) Absence of Otx-2 expression in ATRA-treated Xenopus embryos. (F) Otx-2 is expressed in DN RARb-expressing ATRA-treated embryos. Anterior is to the left; embryo in D faces upwards. ATRA treatment was from stage 8 until stage 13.
	Fig. 3. (A) DN RARb-inhibited transactivation of a DR5 reporter construct can be alleviated by co-injection of RAR RNA, not RXR RNA, indicating that DN RARb specifically inhibits activities of endogenous RARs and that it does not squelch RXRs (or other cofactors). The reporter DNA (165 pg of -1470+156Luc; RARb promoter) was co-injected with 2 ng hRARa RNA (DN + RAR), or 2 ng hRXRa RNA (DN + RXR), and/or 0.5 ng DN RARb RNA. (B) Two synthetic RXR-selective ligands, SR11246 and HX600, are not teratogenic for Xenopus embryos, in contrast to 9-cis-RA. Treatment with retinoid ligands was from stage 10 till stage 12. (C) 9-cis-RA, SR11246, and HX600 penetrate Xenopus embryos and activate the ligandbinding domain of xRXRb. Zygotes were injected with a mixture of 55 pg Gal4-xRXR(DE) and 165 pg Gal-Luc, treated from stage 10 with the indicated retinoids for 7 hours, and assayed for luciferase activity. Bars represent means ± s.e.m. of 2 pools of 10 embryos. no, no ligand (value set to 1); 9-cis, 9-cis-RA; SR, SR11246; HX, HX600. Similar results were obtained in three independent experiments.
	Fig. 4. DN RARb expression causes morphological changes in the hindbrain of Xenopus embryos. CSLM images of fluorescent CNSs of Xenopus stage 47-tadpoles labeled with anti-neural antibodies 2G9 and Xen-1. (A) CNS of control (GR injected) embryo. Clearly visible are the forebrain (pronounced telencephalic olfactory bulbs (olf) connecting to the olfactory placodes), the optic lobes (opt) of the midbrain, and the individual rhombomeres of the hindbrain (hb) (r1-6 are separated, whereas r7 and r8 form one unit). (B,C) CNS of tadpoles injected with 1.75 ng DN RARb RNA before the first cleavage. A relatively normal fore- and midbrain are accompanied by a severely malformed hindbrain. ov, otic vesicle; sc, spinal cord.
	Fig. 5. DN RARb anteriorises rhombomeres 5 and 6. Flat-mounted hindbrains of GR- (A) or DN RARb-expressing embryos (B,C) in which the reticulospinal neurons have been retrogradely labeled with fluorescein-labeled dextran. Ectopic Mauthner neurons are found in r5 and r6 (large arrows in B,C) and sometimes also in r4 (C). Small white arrows in C point to Mauthner axons crossing the midline.
	Fig. 6. Expression of Krox-20 (A-F), Krox-20 and En-2 (G-J), Hoxb-3 (K-M), and Hoxb-9 (P,Q) in stage-25 control (i.e. unilaterally GR RNA injected) embryos (A,G,K,P) and unilaterally DN RARb-expressing embryos (B-F,H-J,L-M, Q); anterior is to the left. (N) Expression of Otx-2 in a stage-19 GR RNA-injected control embryo and (O)in a DN RARb-expressing embryo; anterior is towards the reader. White arrows indicate ectopic expression in the CNS. Black arrowheads point to cells ectopically expressing Krox-20 (C) or Hoxb-3 (I) outside the CNS. Embryos in E-J were cleared with 1:2 benzyl alcohol/benzyl benzoate. Scale bars (in A for A-D; in G for G-J; in K for K-O; in P for P-Q) 100 mm. The embryo in J was stained for b-gal to reveal the expression of coinjected lacZ RNA.
	Fig. 7. Summary of the major results of expression of DN RARb. The left part depicts the control situation for the Xenopus hindbrain: a segmented structure marked by restricted Krox-20 (k) expression domains in r3 and r5, and one Mauthner neuron (M) in r4. DN RARb expression can probably disturb segmentation of the hindbrain and can result in a hindbrain with preferential r3 (k), r4 (M), and r5 (k) identity.